Scavenging Effect of Methanolic Extracts of Peanut Hulls on Free

zolium (NBT), and phenazine methosulfate were purchased from. E. Merck ... 80 % A and 20 % B linearly to 60 % A and 40 % B in 35 min (Yen et al., 1993...
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J. Agric. Food Chem. 1994, 42, 629-632

Scavenging Effect of Methanolic Extracts of Peanut Hulls on Free-Radical and Active-Oxygen Species Gow-Chin Yen'?+ and Pin-Der Duht Department of Food Science, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan, Republic of China, and Department of Food Health, Chia-Nan Junior College of Pharmacy, Tainan, Taiwan, Republic of China

The scavenging effect of methanolic extracts of peanut hulls (MEPH) on free-radical and active-oxygen species was investigated. MEPH showed marked activity as a radical scavenger in the experiment using l,l-diphenyl-2-picrylhydrazyl radical, indicating that MEPH has effective activities as a hydrogen donor and as a primary antioxidant to react with lipid radicals. MEPH also possessed antioxidative activity toward hydrogen peroxide (H202)and superoxide ( 0 2 9 , indicating that MEPH has a scavenging activity on H2Oz and OZ'-. The scavenging effect of MEPH on hydroxyl radical was investigated by means of electron paramagnetic resonance spectrometry. MEPH exhibited a marked scavenging effect on hydroxyl radical, and the scavenging activity of MEPH depended on its concentrations. These results indicate that MEPH is also active as an oxygen scavenger and as a secondary antioxidant. The overall antioxidant effect of MEPH on lipid peroxidation might be attributed to ita properties of scavenging free-radical and active-oxygen species.

INTRODUCTION Oxidative deterioration readily occurs spontaneously when materials containing lipids or lipid-containing foods are exposed to air. Oxidation interferes seriously with the efficiency of the processing steps: it can result in organoleptic rancidity in the finished products, making them unacceptable to consumers, and it can cause other degrading effects such as vitamin destruction, nutritional losses, and discoloration (Sherwin, 1978). The addition of antioxidants to foods is effective in retarding the oxidation of fats. The possible toxicity of synthetic chemicals used as antioxidants has been long guestioned (Imaida et al., 19831,stimulating investigation of the effectiveness of naturally occurring compounds with antioxidative properties (Wu et al., 1982). Antioxidants are classified into four types according to the mechanism of action: chain breaker (or free-radical inhibitor), peroxide decomposer, metal inactivator, or oxygen scavenger (Yagi, 1970; Dziezak, 1986). Much literature on the mechanism of action of antioxidants is available (Chimi et al., 1991;Okamoto et al., 1992). For example, Hayase et al. (1989)speculated that melanoidin showed a greater rate of scavenging hydroxyl radicals as a result of containing reductones, enamines, or pyrrolelike structures. The inhibition of autoxidation by hydroxytyrosol may be related to the ability to scavenge the peroxyl radical (Chimi et al., 1991). Although the investigation of methanolic extracts of peanut hulls (MEPH) on antioxidative activity is reported (Duh et al., 1992;Yen et al., 1993;Yen and Duh, 19931, there is nothing on the mechanism of action of the antioxidative activity by MEPH. Thus, the objective of our work was to elucidate the mechanism of action of the antioxidative effect by MEPH. MATERIALS AND METHODS Chemicals. Catechin, dihydronicotinamidadenine dinucleotide (NADH), butylated hydroxyanisole (BHA), and 5,5dimethyl-l-pyrrolineN-oxide (DMPO)were obtained from Sigma

* Author to whom correspondence should be addressed. f

t

National Chung Hsing University. Chia-Nan Junior Colleg of Pharmacy.

Chemical Co. (St. Louis, MO). Hydrogen peroxide,tetramethyl murexide (TMM), hexamethylenetetramine, nitro blue tetrazolium (NBT),and phenazinemethosulfate were purchased from (DPPH) E. Merck (Germany). l,l-Diphenyl-2-picrylhydrazyl was obtained from Fluka Chemie AG (Switzerland). Materials. Peanuts of Tainan select no. 11, Spanish type, were obtained from the Tainan District Agriculture Improvement Station, Taiwan, Republic of China. After harvest, the peanuts were hand-shelled. Peanut hulls were ground into a fine powder in a mill (Tecator Cemotec 1090sample mill, Hoganas,Sweden), sealed in a plastic bottle, and stored at 4 "C until used. Extractions. Peanut hull powder (5.0 g) was extracted with methanol (50 mL) overnight in a shaking incubator at ambient temperature. The extracts were filtered; the residue was reextracted under the same conditions. The combined filtrates were evaporated in vacuum below 40 "C on a rotary evaporator to a final volume of 5 mL. The concentration of the extracts in the solvent was 44.5 f 1.98 mg/mL, which was the averge of triplicate analyses. Quantitative Analysis of Luteolin. The luteolin in peanut hulls was determined by HPLC performed with a Hitachi liquid chromatograph (Hitachi, Ltd., Tokyo) consisting of a Model L-6200 pump, a Rheodyne Model 7125 syringe loading sample injector, a Model L-4200 UV-vis detector set at 254 nm, and a Model D-2500 integrator. A LiChrosphere 100 RP-18 reversedphase column (5 pm, 125 X 4 mm id., E. Merck) was used for analysis. The volume injected was 10 pL. The elution solvents were water-acetic acid (99:l v/v) (A) and methanol (B). The gradient elution program was set at 1.6 mL/min, starting with 80 % A and 20 % B linearly to 60 % A and 40 % B in 35 min (Yen et al., 1993). To determine the luteolin in the methanolic extracts, an authenic sample of luteolin was prepared; dilutions were made so that the range of concentration correlated with the estimated content of luteolin in the samples. The content of luteolin in the methanolic extracts of peanut hulls was calculated from the standard curve of luteolin. Triplicate samples were run for each set. Determination of Total Phenolic Compounds. The total phenoliccompoundspresent in the peanut hulls was determined spectrophotometricallyusing Folin-Denis reagent (AOAC,19%). The methanolic extract (0.1 mL) of peanut hulls in a volumetric flask was diluted with glass-distilledwater (75 mL). Folin-Denis reagent (5 mL) was added, and the contents of the flask were mixed thoroughly. After 3 min, NazCOs solution (10 mL; concentration 10 g/100 mL) was added and finally quantified to 100 mL with glass-distilled water; the mixture was allowed to stand

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for 30 min with intermittent shaking. The blue color was measured with a spectrophometer (Hitachi U-2OOO). The concentration of total phenolic compounds in the peanut hulls was determined by comparison with the absorbance of standard catechin at different concentrations. Determination of the Effects on DPPH Radical. Four milliliters of methanolic extracts of peanut hulls (final concentration 1.92 mg/mL) decolorized with cartridges (Sep-Pak CIS, Waters) was added to a methanolic solution (1 mL) of DPPH radical (final concentration of DPPH was 2.0 X lo-' M). The mixture was shaken vigorously and left to stand for 30 min; the absorbance of the resulting solution was measured at 517 nm with a Spectrophotometer (Hitachi U-2OOO) (Blois, 1958). All tests and analyses were run in three replicates and averaged. Measurements of Chelating Activity on Metal Ions. The chelating activity of MEPH on Fez+ and Cu2+was measured according to the method of Shimada et al. (1992). To prevent the color of MEPH from interferingwith spectrometricalanalysis, MEPH was decolorized with cartridges (Sep-Pak C18, Waters); the final concentration was 0.192 mg/mL. No contamination of Fe and Cu ions was found in the test solution determined by inductivelycoupled plasma mass spectrometer (ICP-MS,PerkinElmer Elan 5000). Decolorized solution (2.0 mL) was added to hexamine buffer (2.0 mL, 10 mM) containing KCl(10 mM) and FeSoc7HzO (3mM) or CuSO&HzO (3mM), and then tetramethyl murexide [0.2 mL, 1 mM (TMM)] was added. Absorbance at 480 nm was measured withaspectrophotometer (HitachiU-2OOO). The measurement was carried out at 20 "C to prevent oxidation of Fez+. Determination of the Effects on Hydrogen Peroxide. The ability of MEPH to scavenge hydrogen peroxide was determined spectrophotometrically (Ruch et al., 1989). A solution (2 mM) of hydrogen peroxide was prepared in phosphate-buffered saline [ (PBS) pH 7.41 at 20 "C. Hydrogen peroxide concentration was determined spectrophotometrically at 230 nm using a molar extinction coefficient for hydrogen peroxideof 81M-' cm-l (Beers and Sizer, 1952). Methanolicextracts of peanut hulls decolorized with cartridges (Sep-Pak C18, Waters) were addedtothe hydrogen peroxide solution at final concentrations of 0, 0.06,0.6, or 1.2 mg/mL at 20 "C. Absorbance of hydrogen peroxide at 230 nm was determined 10 min later in a spectrophotometer (Hitachi U-2000) against a blank solution containing MEPH (0,0.06,0.6, or 1.2 mg/mL) in PBS without hydrogen peroxide. All testa and analyses were run in replicates and averaged. Determination of the Effects on Superoxide. Superoxide was determined by means of spectrophotometric measurement of the product on the reduction of nitro blue tetrazolium (Nishikimi et al., 1972). Superoxide was generated in a nonenzymic (phenazine methosulfateNADH) system (Nishikimi et al., 1972). The nonenzymic generation of superoxide was measured in samples that contained MEPH (0.48 mg/mL) decolorized with cartridges (Sep-Pak C18, Waters), phenazine methosulfate (20 pM), NADH (156 pM), and nitro blue tetrazolium (50 rM) in phosphate buffer (0.1 M, pH 7.4). After 5 min of incubation at ambient temperature, the color was read at 560 nm against blank samples. All testa and analyses were run in replicates and averaged. Detection of Hydroxyl Radical by EPR Spectrometry. The hydroxyl radical rapidly reacts with the nitrone spin trap 5,5-dimethylpyrrolidine N-oxide (DMPO) (Sigma), and the resultant DMPO-OH adduct is detected by electron paramagnetic resonance (EPR) spectrometry (Rosen and Rauckman, 1984). The EPR spectrum was recorded 2 min after MEPH (0.5 mL) was mixed with HzOz (0.2 mL, 10 mM), Fez+ (0.2 mL, 10 mM), and DMPO (0.2 mL, 0.3 M) using an EPR spectrometer (Bruker ER 200 D 10/12)set at the following conditions: 3480-G magnetic field, 1.0-G modulation amplitude, 0.5-8time constant, and 200-s scan period (Shi and Dalal, 1991). RESULTS AND DISCUSSION

Chemical Characteristics of MEPH. The contents of luteolin and total phenolic compounds in MEPH were 2.58 and 7.70 mg/g of hulls, respectively. The high content of total phenolic compounds in MEPH is responsible for inhibition of lipid peroxidation (Yen et al., 1993). Since the lipid peroxidation is induced and developed by free

Table 1. Scavenging Effects of Methanolic Extracts of Peanut Hulls (MEPH) and Other Compounds on l,l-Diphenyl-2-picrylhydrazyl Radical comM10 absorbance at 517 nm* inhibitionb ( % ) control 0.224 f 0.0087A oc MEPH 0.024 f 0.0036B 89.3 f 1.65B catechin 0.016 0.0003C 92.7 f 1.04A BHA 0.017 f 0.0016C 92.6 0.67A a The concentrations of MEPH, catechin, and butylated hydroxyanisole (BHA) were 1.5 mg/mL, 8 pM, and 240 pM, respectively. b Each value is the mean f standard deviation of three replicate analyses. Values within a column with the same upper case letters are not significantly different at p > 0.05.

*

*

Table 2. Chelating Effect of Different Compounds on Fd+ and CUI+ absorbance*(480 nm) compda Fez+ CuZ+ control 0.778 h 0.001A 1.047 0.005A MEPH 0.773 0.008A 1.042 f 0.012A citric acid 0.484 0.001B 0.968f 0.001B EDTA 0.230 f 0.002C 0.370 t 0.012C OThe concentrations of methanolic extracts of peanut hulle (MEPH),citric acid, and ethylenediaminetetraaceticdieodium salt dihydrate (EDTA) were O.O96/mL, 0.5 M, and 0.5 M, respectively. Each value is the mean f standard deviation of three replicate analyses. Values within a column with the same upper case letters are not significantly different at p > 0.05.

*

*

radicals and active oxygens (Sherwin, 1978;Namiki, 1990), free-radical scavengers and oxygen quenchers may inhibit lipid peroxidation (Tore1et al., 1986). Phenolic compounds that exhibit scavenging efficiency on free radicals and active oxygens are numerous and widely distributed within the plant kingdom (Chimi et al., 1991; Namiki, 1990).Due to the high content of phenolic compounds found in MEPH, therefore, the investigation of the reactivity of MEPH with free radicals or with active oxygens will be helpful in understanding the mechanism of antioxidative function of MEPH. Measurement of Radical-ScavengingActivity. The scavenging activity of MEPH on the DPPH radical is shown in Table 1. Methanolic extracts of peanut hulls decolorized with cartridges (Sep-Pak C18, Waters) at a concentration 1.5 mg/mL exhibited about 90 9% scavenging activity. Hence, methanolic extracts of peanut hulls mixed with DPPH decolorized DPPH due to its hydrogendonating ability (Blois, 1958; Shimada et al., 1992). Catechin and butylated hydroxyanisole (BHA) at concentrations of 8 and 240 pM, respectively, also exhibited marked scavenging activity (>go%). These results show that MEPH is a free-radical inhibitor, a primary antioxidant that reacts with free radicals, particularly the hydroperoxide radical, which is the major propagator of the chain autoxidation of fats, so breaking the chain (Lea, 1958). M e a s u r e m e n t of Chelating Activity on Metal Ions. Although chelating agents are not antioxidants, they play a valuable role in the stabilization of fatty foods against rancidity. The ability of MEPH to form complexes with metal ions is demonstrated in Table 2. EDTA and citric acid showed chelating effect, but decolorized MEPH showed no chelating activity on metal ions. Luteolin containing 3',4'-dihydroxy, 5,7-dihydroxy,and 4-CO groups was identified as an antioxidative component from MEPH (Duh e t al.,1992)and reported to form a ligand with copper ion (Hudson and Lewis, 1984). However, in the present work, MEPH may lose chelating components as a result of decolorizing treatment by cartridges (Sep-Pak CIS, Waters), and it may be the major cause for suppression of chelating metal ions.

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Table 3. Scavenging Effects of Methanolic Extracts of Peanut Hulls (MEPH), Butylated Hydroxyanisole (BHA), and Catechin on Hydrogen Peroxide hydrogen peroxide" compd concn (mM) inhibition" (%) MEPH 0 (mg/mL) 2.00A OE 1.98 f 0.07AB 1.0 f 3.50E 0.06 (mg/mL) 41.8 f 2.36B 0.60 (mg/mL) 1.17 f 0.05D 1.20 (mg/mL) 0.22 f 0.04E 89.1 f 1.88A 8.7 f 1.16D 43(pM) 1.89 f 0.08B BHA 13.8 f 4.16C 1.72 f 0.08C catechin 14 (pM) 0 Each value is the mean f standard deviation of three replicate analyses. Values within a column with the same upper case letters are not significantly different at p > 0.05.

Table 4. Influence of Investigated Compounds on Superoxide Generation by Phenazine Methosulfate and NADH. absorbance at absorbance at compdb 560 nmc compdb 560 nmc BHA 0.244 f 0.009B control 0.331 f 0.039A 0.178 f 0.013C 0.248 f 0.016B MEPH catechin Samples containing phenazine methosulfate (20 pM), NADH (156 pM), and nitro blue tetrazolium (50 pM) in 0.1 M phosphate buffer (pH 7.4) were incubated for 5 min at room temperature and read at 560nm. Theconcentrationsof methanolicextracta of peanut hulls (MEPH),butylated hydroxyanisole (BHA), and catechin were 0.48 mg/mL, 76 pM, and 2.5 pM, respectively. Each value is the mean f standard deviation of six replicate analyses. Values within a column with the same upper case letters are not significantly different at p > 0.05.

Scavenging of Hydrogen Peroxide by MEPH. The scavenging activity of MEPH on hydrogen peroxide is shown in Table 3. MEPH was capable of scavenging hydrogen peroxide in a concentration-dependentfashion after 10 min of incubation. With MEPH (1.2 mg/mL), the concentration of hydrogen peroxide was diminished from 2 to 0.22 mM, representing a hydrogen peroxide reduction to 1.4833 pmol of HaOdmg of MEPH. Namiki (1990) indicated that hydrogen peroxide has only a weak activity to initiate lipid peroxidation, but its activity as an active-oxygen species comes from its potential to produce the highly reactive hydroxyl radical through the Fenton reaction (Cohen and Heikkila, 1974). Therefore, the ability of MEPH to scavenge hydrogen peroxide may contribute to inhibition of the peroxidation of lipid. Scavenging of Superoxide by MEPH. The ability of MEPH to scavenge superoxide generated by phenazine methosulfate-NADH (PMS-NADH) is shown in Table 4. The addition of MEPH (0.48mg/mL) to the PMS-NADH system significantly (p < 0.05) diminished production of superoxide. Catechin (2.5 pM) and BHA (75 pM) also inhibited superoxide. Ilan et al. (1976) noted that superoxide acta as an oxidizing or reducing agent, depending on the substrate oxidation potential. Superoxide also decomposes to form stronger oxidative species such as singlet oxygen, hydroxyl radical, and hydrogen peroxide that initiate the peroxidation of lipid (Dah1 and Richardson, 1978). Superoxide indirectly initiates lipid oxidation as a result of superoxide and hydrogen peroxide that serve as precursors of singlet oxygen and hydroxyl radicals (Kellogg and Fridovich,l975; Aurand et al., 1977). Some reseachen (Lavelleet al., 1973;Tyler, 1975;Goldstein and Weissmann, 1977) reported that superoxide directly initiated lipid peroxidation. Robak and Gryglewski (1988) reported that antioxidant properties of flavonoids are effected mainly via scavenging of superoxide; the marked antioxidant activity of MEPH may be concerned with its higher scavenging of superoxide. Scavenging of Hydroxyl Radical by MEPH. To investigate the reaction of hydroxyl radical, the well-known

C

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mg MEPH

Figure 1. Scavenging of hydroxyl radical by methanolic extracts of peanut hulls (MEPH): (a) EPR spectrum recorded 2.5 min after mixing in a phosphate buffer solution (pH 7.2) of 0.055 M DMPO with 1.82 mM Fez+and 1.82 mM HzOz; (b) as for (a), but with 24.0 mg of MEPH; (c) as for (a), but with 14.4 mg of MEPH; (d) as for (a), but with 4.8 mg of MEPH. Spectrometer settings: receiver gain, 8 X 10; modulation amplitude, 1.0 G; scan time, 200 s; field, 3480 i 50 G; time constant, 0.5 s. Table 5. Effect of Methanolic Extracts of Peanut Hulls (MEPH) on EPR Signal Intensity of DMPO-OH Spin Adduct. re1 EPR re1 EPR signal signal amt (mg) intensity amt (mg) intensity control 100.0 14.4 46.6 4.8 70.1 24.0 28.8 a Treatment and other details are the same aa for Figure 1.

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Fenton reaction (Fe2++ H202 Fe3++ OH- + 'OH) was the source of hydroxyl radical. Figure 1 shows that the reaction of Fe2+with H202 in the presence of spin trapping agent DMPO generated a 1:2:2:1 quartet with hyperfine coupling parameters (anand ah = 14.9 G). According to Figure lb, MEPH (24.0mg) in the reaction system of Figure l a markedly scavenges the DMPO-OH spin adduct. Table 5 shows the EPR signal intensity of DMPO spin adducta formed by Fenton reaction. Methanolic extracts of peanut hulls a t amounts of 4.8,14.4, and 24.0 mg scavenged 29.9, 53.4, and 71.2% of hydroxyl radicals, respectively. Apparently, the activity to scavenge hydroxyl radicals by MEPH that we tested depended upon ita concentrations. The hydroxyl radical is an extremely reactive free radical formed in biological systems (Hochstein and Atallah, 1988) and reacts rapidly with molecules of almost every type found in living cells, such as sugars, amino acids, phospholipids, DNA bases, and organic acid (Halliwell and Gutteridge, 1984). The hydroxyl radical found in biological systems is involved as an active intermediate in physiological processes (Czapski, 1984). Hydroxyl radicals are known to be capable of abstracting hydrogen atoms from membrane lipids (Fong et al., 1973) and bring about peroxidic reactions of lipids (Kitada et al., 1979). Methanolic extracts of peanut hulls have been demonstrated to exhibit markedly antioxidative activity (Duh et al., 1992). Apparently, the ability to quench the hydroxyl radical by

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MEPH as we tested seems t o relate directly t o prevention of propagation of t h e process of lipid peroxidation. Husain e t al. (1987) reported that t h e overall antioxidant effect of flavonoids on lipid peroxidation is t h e result of scavenging of hydroxyl radicals and superoxide anions at t h e stage of initiation and termination of peroxyl radicals. BHA and butylated hydroxytoluene function as antioxidants due to termination of reactions of free radicals of t h e chain type t h a t lead t o lipid peroxidation, whereas citric acid functions via quenching of heavy-metal-catalyzed autoxidation (Dziezak, 1986). The antioxidative properties of rosemary oleoresin in sausages are attributed t o termination of free-radical reactions a n d quenching of reactive oxygen species (Richardson a n d Dahl, 1983). In conclusion, we demonstrated methanolic extracts of peanut hulls have a strong hydrogen-donating ability a n d are good scavengers of active oxygen species, including hydroxyl radical, superoxide, a n d hydrogen peroxide. This property seems t o be important in explaining how t h e antioxidative activity of MEPH arises. ACKNOWLEDGMENT This research work was partially supported by t h e National Science Council, Republic of China, under Grant NSC 82-0409-B005-108. LITERATURE CITED AOAC. Official Methods of Analysis, 14th ed.; Association of Official Analytical Chemists: Washington, DC, 1984; pp 187188. Aurand, L. W.; Boonme, N. H.; Giddings, G. G. Superoxide and singlet oxygen in milk lipid peroxidation. J.Dairy Sei. 1977, 60, 363-369. Beers, R. F.; Sizer, I. W. A spectrofluorometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 1952, 195, 133-140. Blois, M. S. Antioxidant determination by the use of a stable free radical. Nature 1958,4617, 1198-1200. Chimi, H.; Cillard, J.; Cillard, P.; Rahmani, M. Peroxyl and hydroxyl radical scavenging activity of some natural phenolic antioxidants. J. Am. Oil Chem. SOC.1991, 68, 307-312. Cohen, G.; Heikkila, R. E. The generation of hydrogen peroxide superoxide radical and hydroxyl radical by P-hydroxydopamine, dialuric acid, and related cytotoxicagents. J.Biol. Chem. 1974,249, 2447-2452. Czapski,G. Reaction of 'OH. Methods Enzymol. 1984,105,209214. Dahl, M. K.; Richardson,T. Photogeneration of superoxideanion in serum of bovine milk and in model systems containing riboflavin and amino acids. J. Dairy Sei. 1978,61, 400-407. Duh, P. D.; Yeh, D. 5.; Yen, G. C. Extraction and identification of an antioxidative component from peanut hulls. J. Am. Oil Chem. Soc. 1992,69, 814-818. Dziezak, J. D. Preservatives: Antioxidants. Food Technol. 1986, 40, 94-102. Fong,K. L.; McCay, P. B.; Poyer, J. L. Evidencethat peroxidation of lysosomal membrane is initiated by hydroxy radicals produced during flavin enzyme activity. J.Biol. Chem. 1973, 248, 7792-7797. Goldstein, I. M.; Weissmann, G. Effects of the generation of superoxide on permeability of liposomes. Biochem. Biophys. Res. Commun. 1977, 75, 604-609. Halliwell, B.; Gutteridge, M. C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 1984, 219, 1-14. Hayase, F.; Hirashima, S.; Okamota, G.; Kato, H. Scavenging of active oxygens by melanoidins. Agric. Biol. Chem. 1989, 53, 3383-3385. Hochstein, P.; Atallah, A. S. The nature of the oxidants and antioxidant systems in the inhibition of mutation and cancer. Mutat. Res. 1988,202, 363-375. Hudson, B. J.; Lewis, J. I. Polyhydroxy flavonoid antioxidants for edible Oils. Structure criteria for activity. Food Chem. 1983,10, 47-55.

Yen and Duh Husain, S. R.; Cillard, J.; Cillard, P. Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 1987,26, 2489-2491. Ilan, Y. A.; Gzapski, G.; Meisel, D. The one-electron transfer redox potential of free radical. I. The oxygen/superoxide system. Biochim. Biophys. Acta 1976, 430, 209-224. Imaida, K.; Fukushima, S.; Shirai, T.; Ohtani, M.; Nakanish, K.; Ito, N. Promoting activities of butylated hydroxyanisoleand butylated hydroxytolueneon 2-stage urinary bladder carcinogenesis and inhibition of y-glutamyl transpeptidaae-positive foci development in the liver of rats. Carcinogenesis1983,4, 895-899. Kellogg, E. W., 111; Fridovich, I. Superoxide, hydrogen peroxide and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J. Biol. Chem. 1975,250,8812-8817. Kitada, M.; Igarashi, K.; Hirose, S.; Kitagawa, H. Inhibition by polyamines of lipid peroxideformation in rat liver microsomes. Biochem. Biophys. Res. Commun. 1979,87, 388-394. Lavelle,F.; Michelson,A. M.; Dimitrijevic, L. Biologicalprotection by superoxide dismutase. Biochem. Biophys. Res. Commun. 1973,55, 350-357. Lea, C. H. Some nutritional and allied problems confronting the food manufacturer: Technologicalaspects of antioxidants. J. Sci. Food Agric. 1958, 9, 621-632. Namiki,M. Antioxidants/antimutagensin Foods. Crit.Rev.Food Sci. Nutr. 1990, 29 (4), 273-300. Nishikimi, M.; Rao, N. A.; Yagi, K. The occurrenceof superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 1972, 46,849-853. Okamato, G.; Hayase, F.; Kato, H. Scavenging of active oxygen species by glycated proteins. Biosci., Biotechnol., Biochem. 1992,56, 928-931. Richardson, T.; Dahl, M. K. Lipid Oxidation. In Deuelopment in Dairy Chemistry;Fox, F., Ed.; Applied Science Publishing: Barking, England, 1983; p 309. Robak, J.; Gryglewski, R. J. Flavonoids are scavengers of superoxide anions. Biochem. Pharmacol. 1988,37,837-841. Rosen, G. M.; Rauckman, E. J. Spin trapping of superoxide and hydroxyl radicals. Methods Enzymol. 1984,105, 198-209. Ruch, K. J.; Cheng,S.-J.;Klaunig,J. E. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechinisolated from Chinese green tea. Carcinogensis 1989, 10,1003-1008. Sherwin, E. R. Oxidation and antioxidants in fat and oil processing. J. Am. Oil Chem. SOC.1978,55, 809-814. Shi, X.;Dalal, N. S. Antioxidant behavior of caffeine: Efficient scavenging of hydroxyl radicals. Food Chem. Toxicol. 1991, 29,143. Shimada, K.; Fujikawa, K.; Yahara, K.; Nakamura, T. Antioxidative properites of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 1992,40, 945-948. Torel, J.;Cillard, J.;Cillard, P. Antioxidant activity of flavonoids and reactivity with peroxy radical. Phytochemistry 1986,25, 383-386. Tyler, D. D. Role of superoxide radicals in the lipid peroxidation of intracellular membranes. FEBS Lett. 1975, 51, 180-183. Wu, J. W.; Lee, M. H.; Ho, C. T.; Chang, S. S. Elucidation of the chemical structure of nature antioxidants isolated from rosemary, J. Am. Oil Chem. SOC.1982,59, 339-345. Yagi, K. A rapid method for evaluation of autoxidation and antioxidants. Agric. Biol. Chem. 1970, 34, 142-145. Yen, G. C.; Duh, P. D. Antioxidative properties of methanolic extracts of peanut hulls. J.Am. Oil Chem. SOC.1993,70,383386. Yen, G. C.; Duh, P. D.; Tsai, C. L. Relationship between antioxidant activity and maturity of peanut hulls. J. Agric. Food Chem. 1993,41,67-70. Received for review August 12,1993. Revised manuscript received December 3, 1993. Accepted December 8, 1993.' L¶ Abstract published in Advance ACS Abstracts, February 1, 1994.